Polymorphic form of a tachykinin receptor antagonist

ABSTRACT

This invention is concerned with a novel polymorphic form of the compound 2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine which is a tachykinin receptor antagonist useful in the treatment of inflammatory diseases, pain or migraine, asthma, and emesis. 
     The instant polymorphic form has advantages over the other known forms of 2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine in terms of thermodynamic stability and suitability for inclusion in pharmaceutical formulations.

SUMMARY OF THE INVENTION

This invention is concerned with a novel polymorphic form of thecompound:2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine.

The present invention is also concerned with pharmaceutical formulationscomprising this novel polymorphic form of the compound as an activeingredient and the use of the compound and its formulations in thetreatment of certain disorders.

The novel polymorphic form of this invention is a tachykinin receptorantagonist useful in the treatment of inflammatory diseases, pain ormigraine, asthma, and emesis.

This polymorphic form has advantages over the other known forms of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinein terms of thermodynamic stability and suitability for inclusion inpharmaceutical formulations.

BACKGROUND OF THE INVENTION

Analgesia has historically been achieved in the central nervous systemby opiates and analogs which are addictive, and peripherally bycyclooxygenase inhibitors that have gastric side effects. Substance Pantagonists may induce analgesia both centrally and peripherally. Inaddition, substance P antagonists are inhibitory of neurogenicinflammation.

The neuropeptide receptors for substance P (neurokinin-1; NK-1) arewidely distributed throughout the mammalian nervous system (especiallybrain and spinal ganglia), the circulatory system and peripheral tissues(especially the duodenum and jejunum) and are involved in regulating anumber of diverse biological processes. This includes sensory perceptionof olfaction, vision, audition and pain, movement control, gastricmotility, vasodilation, salivation, and micturition (B. Pernow,Pharmacol. Rev., 35, 85-141 (1983)). The NK1 and NK2 receptor subtypesare implicated in synaptic transmission (Laneuville et al., Life Sci.,42:1295-1305 (1988)).

Substance P is a pharmacologically-active neuropeptide that is producedin mammals and acts as a vasodilator, a depressant, stimulatessalivation and produces increased capillary permeability. It is alsocapable of producing both analgesia and hyperalgesia in animals,depending on dose and pain responsiveness of the animal (see R. C. A.Frederickson et al., Science, 199, 1359 (1978); P. Oehme et al.,Science, 208, 305 (1980)) and plays a role in sensory transmission andpain perception (T. M. Jessell, Advan. Biochem. psyhopharmacol. 28, 189(1981)). For example, substance P is believed to be involved in theneurotransmission of pain sensations [Otsuka et al, "Role of Substance Pas a Sensory Transmitter in Spinal Cord and Sympathetic Ganglia" in 1982Substance P in the Nervous System, Ciba Foundation Symposium 91, 13-34(published by Pitman) and Otsuka and Yanagisawa, "Does Substance P Actas a Pain Transmitter? "TIPS, 8 506-510 (Dec. 1987)], specifically inthe transmission of pain in migraine (see B. E. B. Sandberg et al.,Journal of Medicinal Chemistry, 25, 1009 (1982); M. A. Moskowitz, TrendsPharmacol. Sci., 13, 307-311 (1992)), and in arthritis (Levine, et al.Science, 226 547-549 (1984); M. Lotz, et al., Science, 235, 893-895(1987)). Tachykinins have also been implicated in gastrointestinal (GI)disorders and diseases of the GI tract, such as inflammatory boweldisease [see Mantyh et al., [Neuroscience, 25 (3), 817-37 (1988) and D.Regoli in "Trends in Cluster Headache" Ed. F. Sicuteri et al., ElsevierScientific Publishers, Amsterdam, pp. 85-95 (1987)], and emesis [TrendsPharmacol. Sci., 9, 334-341 (1988), F. D. Tatersall, et al., Eur. J.Pharmacol., 250, R5-R6 (1993)].

It is also hypothesized that there is a neurogenic mechanism forarthritis in which substance P may play a role [Kidd et al., "ANeurogenic Mechanism for Symmetric Arthritis" in The Lancet, 11 Nov.1989 and Gronblad et al., "Neuropeptides in Synovium of Patients withRheumatoid Arthritis and Osteoarthritis" in J. Rheumatol. 15(12) 1807-10(1988)]. Therefore, substance P is believed to be involved in theinflammatory response in diseases such as rheumatoid arthritis andosteoarthritis [O'Byrne et al., Arthritis and Rheumatism, 33 1023-8(1990)].

Evidence for the usefulness of tachykinin receptor antagonists in pain,headache, especially migraine, Alzheimer's disease, multiple sclerosis,attenuation of morphine withdrawal, cardiovascular changes, oedema, suchas oedema caused by thermal injury, chronic inflammatory diseases suchas rheumatoid arthritis, asthma/bronchial hyperreactivity and otherrespiratory diseases including allergic rhinitis, inflammatory diseasesof the gut including ulcerative colitis and Chrohn's disease, ocularinjury and ocular inflammatory diseases, proliferativevitreoretinopathy, irritable bowel syndrome and disorders of bladderfunction including cystitis and bladder detruser hyperreflexia isreviewed in "Tachykinin Receptors and Tachykinin Receptor Antagonists,"C. A. Maggi, R. Patacchini, P. Rovero and A. Giachetti, J. Auton.Pharmacol, 13, 23-93 (1993); see also R. M. Snider, et al., Chem. Ind.,11, 792-794 (1991). Neurokinin-1 receptor antagonists alone or incombination with bradykinin receptor antagonists may also be useful inthe prevention and treatment of inflammatory conditions in the lowerurinary tract, especially cystitis [Giuliani, et al., J. Urology, 150,1014-1017 (1993)]. Other disease areas where tachykinin antagonists arebelieved to be useful are allergic conditions [Hamelet et al., Can. J.Pharmacol. Physiol., 66, 1361-7 (1988)], immunoregulation [Lotz, et al.,Science, 241 1218-21 (1988), Kimball, et al., J. Immunol., 141 (10)3564-9 (1988); A. Perianin, et al., Biochem. Biophys. Res Commun.161,520 (1989)], post-operative pain and nausea [C. Bountra, et al.,Eur. J. Pharmacol., 249, R3-R4 (1993), F. D. Tattersall, et al.,Neuropharmacology, 33, 259-260 (1994)], vasodilation, bronchospasm,reflex or neuronal control of the viscera [Mantyh et al., PNAS, 85,3235-9 (1988)] and, possibly by arresting or slowing β-amyloidmediatedneurodegenerative changes [Yankner et al., Science, 250, 279-82 (1990)]in senile dementia of the Alzheimer type, Alzheimer's disease and DownsSyndrome. Substance P may also play a role in demyelinating diseasessuch as multiple sclerosis and amyotrophic lateral sclerosis [J.Luber-Narod, et. al., poster C.I.N.P. XVIIIth Congress, 28th Jun.-2ndJul., 1992], and in disorders of bladder function such as bladderdetrusor hyper-reflexia [Lancet, 16th May 1992, 1239]. Antagonistsselective for the neurokinin-1 (NK-1) and/or the neurokinin-2 (NK-2)receptor may be useful in the treatment of asthmatic disease (Frossardet al., Life Sci., 49, 1941-1953 (1991); Advenier, et al., Biochem.Biophys. Res. Comm., 184(3), 1418-1424 (1992); P. Barnes, et al., TrendsPharmacol. Sci., 11,185-189 (1993)). Tachykinin antagonists may also beuseful in the treatment of small cell carcinomas, in particular smallcell lung cancer (SCLC) [Langdon et al., Cancer Research, 52, 4554-7(1992)].

It has furthermore been suggested that tachykinin receptor antagonistshave utility in the following disorders: depression, dysthymicdisorders, chronic obstructive airways disease, hypersensitivitydisorders such as poison ivy, vasospastic diseases such as angina andReynauld's disease, fibrosing and collagen diseases such as sclerodermaand eosinophillic fascioliasis, reflex sympathetic dystrophy such asshoulder/hand syndrome, addiction disorders such as alcoholism, stressrelated somatic disorders, neuropathy, neuralgia, disorder related toimmune enhancement or suppression such as systemic lupus erythmatosus(EPO Publication No. 0,436,334), ophthalmic diseases such asconjunctivitis, vernal conjunctivitis, and the like, and cutaneousdiseases such as contact dermatitis, atopic dermatitis, urticaria, andother eczematoid dermatitis (EPO Publication No. 0,394,989).

Substance P antagonists may be useful in mediating neurogenic mucussecretion in mammalian airways and hence provide treatment andsymptomatic relief in diseases characterized by mucus secretion, inparticular, cystic fibrosis [S. Ramnarine, et al., abstract presented at1993 ALA/ATS Int'l Conference, 16-19 May, 1993, published in Am. Rev. ofRespiratory Dis., May 1993].

In the recent past, some attempts have been made to provide peptide-likesubstances that are antagonists for the receptors of substance P andother tachykinin peptides in order to more effectively treat the variousdisorders and diseases mentioned above. For example Lowe, Drugs of theFuture, 17 (12) 1115-1121 (1992) and EPO Publication Nos. 0,347,802,0,401,177 and 0,412,452 disclose various peptides as neurokinin Aantagonists. Also, PCT Patent Publication WO 93/14113 discloses certainpeptides as tachykinin antagonists. In addition, EPO Publication No.0,336,230 discloses heptapeptides which are substance P antagonistsuseful in the treatment of asthma. Merck U.S. Pat. No. 4,680,283 alsodiscloses peptidal analogs of substance P. Certain inhibitors oftachykinins have been described in U.S. Pat. No. 0 4,501,733, byreplacing residues in substance P sequence by Trp residues. A furtherclass of tachykinin receptor antagonists, comprising a monomeric ordimeric hexa- or heptapeptide unit in linear or cyclic form, isdescribed in GB-A-2216529.

The peptide-like nature of such substances make them too labile from ametabolic point of view to serve as practical therapeutic agents in thetreatment of disease. The non-peptidic antagonists of the presentinvention, on the other hand, do not possess this drawback, as they areexpected to be more stable from a metabolic point of view than thepreviously-discussed agents.

It is known that in the central nervous system baclofen[β-(aminoethyl)-4-chlorobenzenepropanoic acid] effectively blocks theexcitatory activity of substance P, but because in many areas theexcitatory responses to other compounds such as acetylcholine andglutamate are inhibited as well, baclofen is not considered a specificsubstance P antagonist. Pfizer WIPO patent applications (PCT PublicationNos. WO 90/05525, WO 90/05729, WO 91/18899, WO 92/12151 and WO 92/12152)and publications (Science, 251,435-437 (1991); Science, 251,437-439(1991); J. Med. Chem., 35, 2591-2600 (1992)) disclose2-arylmethyl-3-substituted amino-quinuclidine derivatives which aredisclosed as being useful as substance P antagonists for treatinggastrointestinal disorders, central nervous system disorders,inflammatory diseases and pain or migraine. A Glaxo European patentapplication (EPO Publication No. 0,360,390) discloses variousspirolactam-substituted amino acids and peptides which are antagonistsor agonists of substance P. A Pfizer WIPO patent application (PCTPublication No. WO 92/06079) discloses fused-ring analogs ofnitrogen-containing nonaromatic heterocycles as useful for the treatmentof diseases mediated by an excess of substance P. A Pfizer WIPO patentapplication (PCT Publication No. WO 92/15585 discloses1-azabicyclo[3.2.2]nonan-3-amine derivatives as substance P antagonists.A Pfizer WIPO patent application (PCT Publication No. WO 93/10073)discloses ethylenediamine derivatives as substance P antagonists. PCTPublication No. WO 93/01169 discloses certain aromatic compounds astachykinin receptor antagonists. A Sanofi publication (Life Sci., 50,PL101-PL106 (1992)) discloses a 4-phenyl piperidine derivative as anantagonist of the neurokinin A (NK2) receptor.

Howson et al. (Biorg. & Med. Chem. Lett., 2 (6), 559-564 (1992))disclose certain 3-amino and 3-oxy quinuclidine compounds and theirbinding to substance P receptors. EPO Publication 0,499,313 disclosescertain 3-oxy and 3-thio azabicyclic compounds as tachykininantagonists. U.S. Pat. No. 3,506,673 discloses certain 3-hydroxyquinuclidine compounds as central nervous system stimulants. A PfizerEPO Patent application (EPO publication 0,436,334) discloses certain3-aminopiperidine compounds as substance P antagonists. U.S. Pat. No.5,064,838 discloses certain 1,4-disubstituted piperidinyl compounds asanalgesics. PCT Publication No. WO 92/12128 discloses certain piperidineand pyrrolidine compounds as analgesics. Peyronel, et al. (Biorg & Med.Chem. Lett., 2 (1), 37-40 (1992)) disclose a fused ring pyrrolidinecompound as a substance P antagonist. EPO Publication No. 0,360,390discloses certain spirolactam derivatives as substance P antagonists.U.S. Pat. No. 4,804,66 discloses certain piperazine compounds asanalgesics. U.S. Pat. No. 4,943,578 discloses certain piperazinecompounds useful in the treatment of pain. PCT Publication No. WO92/01679 discloses certain 1,4-disubstituted piperazines useful in thetreatment of mental disorders in which a dopaminergic deficit isimplicated. PCT Publication No. WO 94/00440 discloses certain morpholinecompounds as substance P antagonists.

Morphological forms of pharmaceutical compounds may be of interest tothose involved in the development of a suitable dosage form because ifthe morphological form is not held constant during clinical andstability studies, the exact dosage used or measured may not becomparable from one lot to the next. Once a pharmaceutical compound isproduces for use, it is important to recognize the morphological formdelivered in each dosage form to assure that the production process usethe same form and that the same amount of drug is included in eachdosage. Therefore, it is imperative to assure that either a singlemorphological form or some known combination of morphological forms ispresent. In addition, certain morphological forms may exhibit enhancedthermodynamic stability and may be more suitable than othermorphological forms for inclusion in pharmaceutical formulations. Asused herein, a polymorphic form of a chemical compound is the samechemical entity, but in a different crystalline arrangement.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a novel polymorphic form of thecompound2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholineand the process for the preparation of this polymorphic form.

The compound2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinehas the structure: ##STR1## and is a tachykinin receptor antagonistuseful in the treatment of inflammatory diseases, pain or migraine,asthma, and emesis.

This particular polymorphic form (herein designated "Form C") hassuperior properties over other crystalline forms of the compound in thatit is thermodynamically more stable than other morphological forms andis more suitable for inclusion in pharmaceutical formulations.

The present invention is also concerned with a process for thepreparation of Form C of2-(S)-(3,5-bis(trifluoromethyl)-benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinewhich comprises:

heating a sample of2-(S)-(3,5-bis(trifluoromethyl)-benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholineof optional morphological composition to a temperature range of 160° to170° C.; and then

returning the sample to ambient temperature.

In particular, the heating of2-(S)-(3,5-bis(trifluoro-methyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinemay be conducted in a differential scanning calorimetric cell in an openpan at a rate of 1° C./minute under a nitrogen atmosphere and the2-(S)-(3,5-bis(trifluoromethyl)-benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinemay be heated to a temperature of 160° C., and then cooled to roomtemperature. Preferably the morphological composition of the starting2-(S)-(3,5-bis(trifluoro-methyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholineis Form B.

In addition, the present invention is concerned with an alternativeprocess particularly useful for the preparation of Form C of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholineon a larger scale comprising:

suspending2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholineof optional morphological composition in water;

adding seed crystals of Form C of2-(S)-(3,5-bis(trifluoro-methyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine;

heating the resultant mixture at 35°-40° C. with stirring for a periodsufficient to result in the formation of Form C of 2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)-methyl)-3-(S)-phenyl-morpholine;and

collecting the resultant Form C of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine.

Similarly, the present invention is also directed to a process for thepreparation of morphologically homogeneous2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine,comprising any of the aforementioned processes.

The compound of this invention, the novel polymorphic form of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine,is a tachykinin receptor antagonists useful in the treatment ofinflammatory diseases, pain or migraine, asthma, and emesis.Accordingly, the present invention is further concerned withpharmaceutical formulations comprising this polymorphic form as anactive ingredient, and the use of this polymorphic form and itsformulations in the treatment of certain disorders.

Form C of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholineis anhydrous and non-hygroscopic and exhibits a high degree of thermalstability as a neat solid and in hydroalcoholic solution.

Form A is an anhydrous crystalline material melting at 254° C. which isobtained directly from recrystallization in the chemical synthesis of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine.

Form B is a hydrated material that is obtained from an aqueoussuspension of Form A at room temperature. Form B is a metastablehydrated form and is converted to Form C either thermally, or by addingForm C seed crystals to a suspension of Form B.

From C may be obtained directly from Form A by seeding an aqueoussuspension of Form A. An aqueous suspension of Form A spontaneouslyconverts to the higher melting polymorph Form C if a few seed crystalsof Form C are present. Form C may also be prepared on a small scale byheating a sample of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholineof optional morphological composition to a temperature range of 160° to170° C., and returning the sample to ambient temperature.

Differential Scanning Calorimeteric Cell [DSC]

Form C of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinewas prepared by heating Form A of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine(prepared as described in Example 1) in a differential scanningcalorimetric (DSC) cell in an open pan to 160° C. at a heating rate of1° C./min under a nitrogen atmosphere.

The DSC curve of Form A at 5° C./min shows onset of a melting endothermat 153° C., followed by a peak melting endotherm at 156° C. The DSCcurve of Form B at 1° C./min shows thermal events due to water loss, amelting endotherm at 143° C., followed by a recrystallization exothermat 147° C., and a melting endotherm at 179° C.

                  TABLE 1                                                         ______________________________________                                        DSC Data [samples heated at a rate of 5° C./min under nitrogen         atmosphere (extrapolated onset temperature)]                                              m.p. (°C.)                                                 ______________________________________                                               Form A 153-155                                                                Form B ca. 145                                                                Form C 176-178                                                         ______________________________________                                    

X-Ray Powder Diffraction (XRPD)

X-Ray powder diffraction studies have been widely used to elucidatemolecular structures, crystallinity and polymorphism. X-ray powderdiffraction (XRPD) patterns were recorded using a Siemens D-5000instrument equipped with a 3 kw X-ray generator (CuKα1 radiation) and aNaI (Ti) scintillation detector. Measurements were made from 3° to 45°(2 theta) with the sample maintained at ambient room temperature.

Form A was characterized by an X-ray powder diffraction pattern withreflections at approximately: 7.7°, 9.5°, 12.7°, 14.0°, 14.5°, 15.3°,15.9°, 17.4°, 18.1°, 19.4°, 20.5°, 22.3°, 22.9°, 23.8°, 24.9°, and 26.5°(2 theta).

Form B was characterized by an X-ray powder diffraction pattern withreflections at approximately: 17.7°, 18.7°, 19.3°, 20.2°, 21.8°, and24.2° (2 theta).

Form C was characterized by an X-ray powder diffraction pattern withreflections at approximately: 11.5°, 12.6°, 15.5°, 16.8°, 20.1°, 21.3°,25.4° and 28.5° (2 theta).

These XRPD patterns confirm that all three samples are distinctcrystalline forms.

Microscopy

Examination of the polymorphic forms was conducted at 10033magnification under plain and polarized light. Forms A and B consistedof irregularly shaped particles with an average size of about 50 μm.Form C was observed to consist of needle shaped particles averagingabout 20-30 μm in length. All the samples appeared birefringent underpolarized light.

Hygroscopicity

The total volatiles content (as established by TGA analysis) of solidsamples of Form A and C upon exposure to various controlled humiditiesis tabulated below.

                  TABLE 2                                                         ______________________________________                                        Total Volatiles Content of Form A and Form C:                                 % RH          Form A   Form C                                                 ______________________________________                                         0            0.2      0.5                                                    11            0.3      0.4                                                    33            0.2      0.4                                                    47            0.2      0.3                                                    76            0.3      0.7                                                    100           0.4      0.9                                                    ______________________________________                                    

This data indicates that both Form A and Form C are essentiallynon-hygroscopic over the entire range of relative humidities. TGA ofForm B reveals a 2.1% weight loss (corresponding to 0.60 moles water)commencing at about 60° C.

Solubility

Form A is soluble to the extent of 5 μg/mL in distilled water at roomtemperature. The aqueous solubility of Form C was determined to be 2.7μg/mL in distilled water at room temperature.

TACHYKININ ANTAGONISM ASSAY

The compound of this invention, the polymorphic Form C of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine,is useful for antagonizing tachykinins, in particular substance P andneurokinin A in the treatment of gastrointestinal disorders, centralnervous system disorders, inflammatory diseases, pain or migraine,asthma, and emesis in a mammal in need of such treatment. This activitymay be demonstrated by the following assay.

A. Receptor Expression in COS

To express the cloned human neurokinin-1 receptor (NK1R) transiently inCOS, the cDNA for the human NK1R was cloned into the expression vectorpCDM9 which was derived from pCDM8 (INVITROGEN) by inserting theampicillin resistance gene (nucleotide 1973 to 2964 from BLUESCRIPT SK+)into the Sac II site. Transfection of 20 ug of the plasmid DNA into 10million COS cells was achieved by electroporation in 800 ul oftransfection buffer (135 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₁₂, 2.4 mM K₂HPO₄, 0.6 mM KH₂ PO₄, 10 mM glucose, 10 mM HEPES pH 7.4) at 260 V and950 uF using the IBI GENEZAPPER (IBI, New Haven, Conn.). The cells wereincubated in 10% fetal calf serum, 2 mM glutamine, 100 U/mlpenicillin-streptomycin, and 90% DMEM media (GIBCO, Grand Island, N.Y.)in 5% CO₂ at 37° C. for three days before the binding assay.

B. Stable Expression in CHO

To establish a stable cell line expressing the cloned human NK1R, thecDNA was subcloned into the vector pRcCMV (INVITROGEN). Transfection of20 ug of the plasmid DNA into CHO cells was achieved by electroporationin 800 ul of transfection buffer suplemented with 0.625 mg/ml Herringsperm DNA at 300 V and 950 uF using the IBI GENEZAPPER (IBI). Thetransfected cells were incubated in CHO media [10 % fetal calf serum,100 U/ml pennicilin-streptomycin, 2 mM glutamine, 1/500hypoxanthine-thymidine (ATCC), 90% IMDM media (JRH BIOSCIENCES, Lenexa,Kans.), 0.7 mg/ml G418 (GIBCO)] in 5% CO₂ at 37° C. until colonies werevisible. Each colony was separated and propagated. The cell clone withthe highest number of human NK1R was selected for subsequentapplications such as drug screening.

C. Assay Protocol using COS or CHO

The binding assay of human NK1R expressed in either COS or CHO cells isbased on the use of ¹²⁵ I-substance P (¹²⁵ I-SP, from DU PONT, Boston,Mass.) as a radioactively labeled ligand which competes with unlabeledsubstance P or any other ligand for binding to the human NK1R. Monolayercell cultures of COS or CHO were dissociated by the non-enzymaticsolution (SPECIALTY MEDIA, Lavallette, N.J.) and resuspended inappropriate volume of the binding buffer (50 mM Tris pH 7.5, 5 mM MnCl₂,150 mM NaCl, 0.04 mg/ml bacitracin, 0,004 mg/ml leupeptin, 0.2 mg/mlBSA, 0.01 mM phosphoramidon) such that 200 ul of the cell suspensionwould give rise to about 10,000 cpm of specific ¹²⁵ I-SP binding(approximately 50,000 to 200,000 cells). In the binding assay, 200 ul ofcells were added to a tube containing 20 ul of 1.5 to 2.5 nM of ¹²⁵ I-SPand 20 ul of unlabeled substance P or any other test compound. The tubeswere incubated at 4° C. or at room temperature for 1 hour with gentleshaking. The bound radioactivity was separated from unboundradioactivity by GF/C filter (BRANDEL, Gaithersburg, Md.) which waspre-wetted with 0.1% polyethylenimine. The filter was washed with 3 mlof wash buffer (50 mM Tris pH 7.5, 5 mM MnCl₂, 150 mM NaCl) three timesand its radioactivity was determined by gamma counter.

The activation of phospholipase C by NK1R may also be measured in CHOcells expressing the human NK1R by determining the accumulation ofinositol monophosphate which is a degradation product of IP₃. CHO cellsare seeded in 12-well plate at 250,000 cells per well. After incubatingin CHO media for 4 days, cells are loaded with 0.025 uCi/ml of ³H-myoinositol by overnight incubation. The extracellular radioactivityis removed by washing with phosphate buffered saline. LiCl is added tothe well at final concentration of 0.1 mM with or without the testcompound, and incubation is continued at 37° C. for 15 min. Substance Pis added to the well at final concentration of 0.3 nM to activate thehuman NK1R. After 30 min of incubation at 37° C., the media is removedand 0.1N HCl is added. Each well is sonicated at 4° C. and extractedwith CHCl₃ /methanol (1:1). The aqueous phase is applied to a 1 ml DowexAG 1×8 ion exchange column. The column is washed with 0.1N formic acidfollowed by 0.025M ammonium formate-0.1N formic acid. The inositolmonophosphate is eluted with 0.2M ammonium formate-0.1N formic acid andquantitated by beta counter.

The compound of the present invention is useful in the prevention andtreatment of a wide variety of clinical conditions which arecharacterized by the presence of an excess of tachykinin, in particularsubstance P, activity. These conditions may include disorders of thecentral nervous system such as anxiety, depression, psychosis andschizophrenia; epilepsy; neurodegenerative disorders such as dementia,including senile dementia of the Alzheimer type, Alzheimer's disease andDown's syndrome; demyelinating diseases such as multiple sclerosis (MS)and amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease) and otherneuropathological disorders such as peripheral neuropathy, for exampleAIDS related neuropathy, diabetic neuropathy, chemotherapy-inducedneuropathy, and postherpetic and other neuralgias; small cell carcinomassuch as small cell lung cancer; respiratory diseases, particularly thoseassociated with excess mucus secretion such as chronic obstructiveairways disease, bronchopneumonia, chronic bronchitis, cystic fibrosis,asthma, and bronchospasm; airways disease modulated by neurogenicinflammation; diseases associated with decreased glandular secretions,including lacrimation, such as Sjogren's syndrome, hyperlipoproteinemiasIV and V, hemochromatosis, sarcoidosis, or amyloidosis; inflammatorydiseases such as inflammatory bowel disease, psoriasis, fibrositis,ocular inflammation, osteoarthritis, rheumatoid arthritis, pruritis, andsunburn; allergies such as eczema and rhinitis; hypersensitivitydisorders such as poison ivy; ophthalmic diseases such asconjunctivitis, vernal conjunctivitis, dry eye syndrome, and the like;ophthalmic conditions associated with cell proliferation such asproliferative vitreoretinopathy; cutaneous diseases such as contactdermatitis, atopic dermatitis, urticaria, and other eczematoiddermatitis; oedema, such as oedema caused by thermal injury; addictiondisorders such as alcoholism; stress related somatic disorders; reflexsympathetic dystrophy such as shoulder/hand syndrome; dysthymicdisorders; adverse immunological reactions such as rejection oftransplanted tissues and disorders related to immune enhancement orsuppression, such as systemic lupus erythmatosus; gastrointestinal (GI)disorders and diseases of the GI tract such as disorders associated withthe neuronal control of viscera such as ulcerative colitis, Crohn'sdisease, irritable bowel syndrome, nausea, and emesis, including acute,delayed, post-operative, late-phase, and anticipatory emesis, such asemesis or nausea induced by for example chemotherapy, radiation,surgery, migraine, toxins, such as metabolic or microbial toxins, viralor bacterial infections, pregnancy, vestibular disorder, motion,mechanical stimulation, gastrointestinal obstruction, reducedgastrointestinal motility, visceral pain, psychological stress ordisturbance, high altitude, weightlessness, opioid analgesics,intoxication, resulting for example from consumption of alcohol, andvariations in intercranial pressure, in particular, for example, drug orradiation induced emesis or post-operative nausea and vomiting;disorders of bladder function such as cystitis, bladder detrusorhyperreflexia, and incontinence; fibrosing and collagen diseases such asscleroderma and eosinophilic fascioliasis; disorders of blood flowcaused by vasodilation and vasospastic diseases such as angina, migraineand Reynaud's disease; and pain or nociception, for example, chronicpain or that attributable to or associated with any of the foregoingconditions especially the transmission of pain in migraine. Hence, thecompound may be readily adapted to therapeutic use for the treatment ofphysiological disorders associated with an excessive stimulation oftachykinin receptors, especially neurokinin-1, and as neurokinin-1antagonists in the control and/or treatment of any of the aforesaidclinical conditions in mammals, including humans.

The compound of the present invention is also of value in the treatmentof a combination of the above conditions, in particular in the treatmentof combined post-operative pain and post-operative nausea and vomiting.

The compound of the present invention is particularly useful in thetreatment of nausea or emesis, including acute, delayed, post-operative,late-phase, and anticipatory emesis, such as emesis or nausea induced byfor example chemotherapy, radiation, surgery, migraine, toxins, such asmetabolic or microbial toxins, viral or bacterial infections, pregnancy,vestibular disorder, motion, mechanical stimulation, gastrointestinalobstruction, reduced gastrointestinal motility, visceral pain,psychological stress or disturbance, high altitude, weightlessness,opioid analgesics, intoxication, resulting for example from consumptionof alcohol, and variations in intercranial pressure. Most especially,this compound is of use in the treatment of emesis induced byantineoplastic (cytotoxic) agents including those routinely used incancer chemotherapy.

Examples of such chemotherapeutic agents include alkylating agents, forexample, nitrogen mustards, ethyleneimine compounds, alkyl sulfonatesand other compounds with an alkylating action such as nitrosoureas,cisplatin, and dacarbazine; antimetabolites, for example, folic acid,purine or pyrimidine antagonists; mitotic inhibitors, for example, vincaalkaloids and derivatives of podophyllotoxin; and cytotoxic antibiotics.

Particular examples of chemotherapeutic agents are described, forexample, by D. J. Stewart in "Nausea and Vomiting: Recent Research andClinical Advances", Eds. J. Kucharczyk, et al., CRC Press Inc., BocaRaton, Fla., USA (1991), pages 177-203, especially page 188. Commonlyused chemotherapeutic agents include cisplatin, dacarbazine (DTIC),dactinomycin, mechlorethamine (nitrogen mustard), streptozocin,cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin(adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine,etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine,bleomycin, and chlorambucil [R. J. Gralla, et al., Cancer TreatmentReports, 68(1), 163-172 (1984)].

The compound of the present invention is also of use in the treatment ofemesis induced by radiation including radiation therapy such as in thetreatment of cancer, or radiation sickness, and in the treatment ofpost-operative nausea and vomiting.

The compound of the present invention are also of use in the preventionor treatment of disorders of the central nervous system such as anxiety,psychosis and schizophrenia; neurodegenerative disorders such as seniledementia of the Alzheimer type, Alzheimer's disease and Down's syndrome;respiratory diseases, particularly those associated with excess mucussecretion, such as chronic obstructive airways disease,broncho-pneumonia, chronic bronchitis, cystic fibrosis and asthma, andbronchospasm; inflammatory diseases such as inflammatory bowel disease,osteoarthritis and rheumatoid arthritis; adverse immunological reactionssuch as rejection of transplanted tissues; gastrointestinal (GI)disorders and diseases of the GI tract such as disorders associated withthe neuronal control of viscera such as ulcerative colitis, Crohn'sdisease and incontinence; disorders of blood flow caused byvasodilation; and pain or nociception, for example, that attributable toor associated with any of the foregoing conditions or the transmissionof pain in migraine (both prophylaxis and acute treatment).

As calcium channel blocking agents the compound of the present inventionis useful in the prevention of treatment of clinical conditions whichbenefit from inhibition of the transfer of calcium ions across theplasma membrane of cells. These include diseases and disorders of theheart and vascular system such as angina pectoris, myocardialinfarction, cardiac arrhythmia, cardiac hypertrophy, cardiac vasospasm,hypertension, cerebrovascular spasm and other ischemic disease.Furthermore, this compound may be capable of lowering elevatedintraocular pressure when administered topically to the hypertensive eyein solution in a suitable ophthalmic vehicle. Also, this compound may beuseful in the reversal of multidrug resistance in tumor cells byenhancing the efficacy of chemotherapeutic agents. In addition, thecompound may have activity in blocking calcium channels in insect brainmembranes and so may be useful as insecticides.

The compound of the present invention is particularly useful in thetreatment of pain or nociception and/or inflammation and disordersassociated therewith such as, for example: neuropathy, such as diabeticor peripheral neuropathy and chemotherapy-induced neuropathy;postherpetic and other neuralgias; asthma; osteoarthritis; rheumatoidarthritis; and especially migraine. The compound of the presentinvention is also particularly useful in the treatment of diseasescharacterized by neurogenic mucus secretion, especially cystic fibrosis.

In the treatment of the clinical conditions noted above, the compound ofthis invention may be utilized in compositions such as tablets, capsulesor elixirs for oral administration, suppositories for rectaladministration, sterile solutions or suspensions for parenteral orintramuscular administration, and the like.

The pharmaceutical compositions of this invention may be used in theform of a pharmaceutical preparation, for example, in solid, semisolidor liquid form, which contains one or more forms of the compound of thepresent invention, as an active ingredient, in admixture with an organicor inorganic carrier or excipient suitable for external, enteral orparenteral applications. The active ingredient may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, solutions, emulsions,suspensions, and any other form suitable for use. The carriers which maybe used are water, glucose, lactose, gum acacia, gelatin, mannitol,starch paste, magnesium trisilicate, talc, corn starch, keratin,colloidal silica, potato starch, urea and other carriers suitable foruse in manufacturing preparations, in solid, semisolid, or liquid form,and in addition auxiliary, stabilizing, thickening and coloring agentsand perfumes may be used. The active object compound is included in thepharmaceutical composition in an amount sufficient to produce thedesired effect upon the process or condition of the disease.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,microcrystalline cellulose, stearic acid, dicalcium phosphate or gums,and other pharmaceutical diluents, to form a solid compositioncontaining a homogeneous mixture of a compound of the present invention,or a non-toxic pharmaceutically acceptable salt thereof. When referringto these mixtures as homogeneous, it is meant that the active ingredientis dispersed evenly throughout the composition so that the compositionmay be readily subdivided into equally effective unit dosage forms suchas tablets, pills and capsules. This solid mixture is then subdividedinto unit dosage forms of the type described above containing from 0.1to about 500 mg of the active ingredient of the present invention. Thetablets or pills of the novel composition may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill may comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components may be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials may be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solution, suitably flavored syrups, and aqueous or oilsuspensions, and similar pharmaceutical vehicles. Suitable dispersing orsuspending agents for aqueous suspensions include synthetic and naturalgums such as tragacanth, acacia, alginate, dextran, sodiumcarboxymethylcellulose, methylcellulose, polyvinylpyrrolidone orgelatin.

Compositions for inhalation or insufflation include suspensions inpharmaceutically acceptable, aqueous or organic solvents, or mixturesthereof, and powders. The liquid or solid compositions may containsuitable pharmaceutically acceptable excipients as set out above.Preferably the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably sterile pharmaceutically acceptable solvents may be nebulizedby use of inert gases. Nebulized suspensions may be breathed directlyfrom the nebulizing device or the nebulizing device may be attached to aface mask, tent or intermittent positive pressure breathing machine.Solution, suspension or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

For the treatment of the clinical conditions and diseases noted above,the compound of this invention may be administered orally, topically,parenterally, by inhalation spray or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. The term parenteral as usedherein includes subcutaneous injections, intravenous, intramuscular,intrasternal injection or infusion techniques.

For the treatment of certain conditions it may be desirable to employthe compound of the present invention in conjunction with anotherpharmacologically active agent. For example, the compound of the presentinvention may be presented together with another therapeutic agent as acombined preparation for simultaneous, separate, or sequential use forthe relief of emesis. Such combined preparations may be, for example, inthe form of a twin pack. A preferred combination comprises the compoundof the present invention with a chemotherapeutic agent such as analkylating agent, antimetabolite, mitotic inhibitor, or cytotoxicantibiotic, as described above. In general, the currently availabledosage forms of the known therapeutic agents for use in suchcombinations will be suitable.

Similarly, for the treatment of respiratory diseases, such as asthma,the compound of the present invention may be used in conjunction with abronchodilator, such as a β₂ -adrenergic receptor agonist or atachykinin antagonist which acts at neurokinin-2 receptors. Also, forthe treatment of conditions that require antagonism of both neurokinin-1and neurokinin-2, including disorders associated withbronchoconstriction and/or plasma extravasation in airways, such asasthma, chronic bronchitis, airways disease, or cystic fibrosis, thecompound of the present invention may be used in conjunction with atachykinin antagonist which acts at neurokinin-2 receptors, or withtachykinin receptor antagonist which acts at both neurokinin-1 andneurokinin-2 receptors. Similarly, for the prevention or treatment ofemesis the compound of the present invention may be used in conjunctionwith other anti-emetic agents, especially 5HT₃ receptor antagonists,such as ondansetron, granisetron, tropisetron, decadron, and zatisetron.Likewise, for the prevention or treatment of migraine the compound ofthe present invention may be used in conjunction with otheranti-migraine agents, such as ergotamines or 5HT₁ agonists, especiallysumatriptan. For the prevention or treatment of inflammatory conditionsin the lower urinary tract, especially cystitis, the compound of thepresent invention may be used in conjunction with an antiinflammatory,such as a bradykinin receptor antagonist. The compound of the presentinvention and the other pharmacologically active agent may beadministered to a patient simultaneously, sequentially or incombination.

The compound of this invention may be administered to patients (animalsand humans) in need of such treatment in dosages that will provideoptimal pharmaceutical efficacy. It will be appreciated that the doserequired for use in any particular application will vary from patient topatient, not only with the particular compound or composition selected,but also with the route of administration, the nature of the conditionbeing treated, the age and condition of the patient, concurrentmedication or special diets then being followed by the patient, andother factors which those skilled in the art will recognize, with theappropriate dosage ultimately being at the discretion of the attendantphysician.

In the treatment of a condition associated with an excess oftachykinins, an appropriate dosage level will generally be about 0.001to 50 mg per kg patient body weight per day which may be administered insingle or multiple doses. Preferably, the dosage level will be about0.01 to about 25 mg/kg per day; more preferably about 0.05 to about 10mg/kg per day. For example, in the treatment of conditions involving theneruotransmission of pain sensations, a suitable dosage level is about0.001 to 25 mg/kg per day, preferably about 0.05 to 10 mg/kg per day,and especially about 0.1 to 5 mg/kg per day. The compound may beadministered on a regimen of 1 to 4 times per day, preferably once ortwice per day. In the treatment of emesis using an injectableformulation, a suitable dosage level is about 0.001 to 10 mg/kg per day,preferably about 0.005 to 5 mg/kg per day, and especially about 0.01 to1 mg/kg per day. The compound may be administered on a regimen of 1 to 4times per day, preferably once or twice per day.

Methods for preparing the polymorphic form of this invention areillustrated in the following Examples. The following examples are givenfor the purpose of illustrating the present invention and shall not beconstrued as being limitations on the scope or spirit of the instantinvention.

EXAMPLE 12-(S)-(3,5-Bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine

N-Methylcarboxy-2-chloroacetamidrazone

Step A: N-Methylcarboxy-2-chloroacetamidrazone

A solution of 5.0 g (66.2 mmol) of chloroacetonitrile in 35 mL of drymethanol was cooled to 0° C. and was treated with 0.105 g (1.9 mmol) ofsodium methoxide. The ice-bath was removed and the mixture was allowedto stir at room temperature for 30 minutes. To the reaction was thenadded 0.110 mL (1.9 mmol) of acetic acid and then 5.8 g (64.9 mmol) ofmethyl hydrazinecarboxylate. After stirring 30 minutes at roomtemperature, the suspension was concentrated in vacuo, and placed on thehigh-vac line overnight, to give 10.5 g (98%) of a yellow powder.

¹ H NMR (CD₃ OD, 400 MHz, ppm): δ3.71 (s, 3H), 4.06 (s, 2H).

Step B: 3,5-Bis(trifluoromethyl)benzyl alcohol,trifluoromethane-sulfonate ester

A solution of 1.00 g (4.1 mmole) of 3,5-bis(trifluoromethyl)benzylalcohol and 1.05 g (5.12 mmole) of 2,6-di-t-butyl-4- methylpyridine in45 mL of dry carbon tetrachloride under a nitrogen atmosphere wastreated with 0.74 mL (4.38 mmole) of trifluoromethanesulfonic anhydrideat room temperature. A white precipitate formed shortly after theaddition of the anhydride. After 90 min, the slurry was filtered undernitrogen with a Schlenk filter, and the filtrate was concentrated invacuo. The residue, which was a two-phase oil, was dissolved undernitrogen in 10 mL of dry toluene. The resulting clear solution was usedimmediately in Step E below.

Step C: N-Benzyl-(S)-phenylglycine

A solution of 1.51 g (10.0 mmol) of (S)-phenylglycine in 5 mL of 2Naqueous sodium hydroxide solution was treated with 1.0 mL (10.0 mmol) ofbenzaldehyde and stirred at room temperature for 20 minutes. Thesolution was diluted with 5 mL of methanol, cooled to 0° C., andcarefully treated with 200 mg (5.3 mmol) of sodium borohydride. Thecooling bath was removed and the reaction mixture was stirred at roomtemperature for 1.5 hours. The reaction was diluted with 20 mL of waterand extracted with 2×25 mL of methylene chloride. The aqueous layer wasacidified with concentrated hydrochloric acid to pH 6 and the solid thatprecipitated was filtered, washed with 50 mL of water, 50 mL of 1:1 v/vmethanol/ethyl ether and 50 mL of ether, and dried to afford 1.83 g(76%) of product, mp 230°-232° C.

Analysis Calcd for C₁₅ H₁₅ NO₂ : C, 74.66; H, 6.27; N, 5.81. Found: C,74.17;H, 6.19; N, 5.86.

Step D: 3-(S)-Phenyl-4-benzyl-2-morpholinone

A mixture of 4.00 g (16.6 mmol) of N-benzyl-(S)-phenylglycine, 5.00 g(36.0 mmol) of potassium carbonate, 10.0 mL of 1,2-dibromoethane and 25mL of N,N-dimethylformamide was stirred at 100° C. for 20 hours. Themixture was cooled and partitioned between 200 mL of ethyl ether and 100mL of water. The layers were separated and the organic layer was washedwith 3×50 mL of water, dried over magnesium sulfate and concentrated invacuo. The residue was purified by flash chromatography on 125 g ofsilica gel eluting with 9:1 v/v, then 4:1 v/v hexanes/ethyl ether toafford 2.41 g (54%) of the product as a solid, mp 98°-100° C.

Mass Spectrum (FAB): m/Z 268 (M+H, 100%). ¹ H NMR (CDCl₃, 200 MHz, ppm):δ2.54-2.68 (m, 1H), 2.96 (dt, J=12.8, 2.8, 1H), 3.14 (d, J=13.3, 1H),3.75 (d, J=13.3, 1H), 4.23 (s, 1H), 4.29-4.37 (m, 1H), 4.53 (dt, J=3.2,11.0), 7.20-7.56 (m, 10H). Analysis Calcd for C₁₇ H₁₇ NO₂ : C, 76.38; H,6.41; N, 5.24. Found: C, 76.06; H, 6.40; N, 5.78.

Step E:4-Benzyl-2-(S)-(3,5-bis(trifluoromethyl)benz-yloxy)-3-(S)-phenylmorpholine

A solution of 0.500 g (1.87 mmole) ofN-benzyl-3-(S)-phenylmorpholin-2-one in 10 mL of dry THF was cooled to-75° C. under nitrogen and was treated dropwise with 2.06 mL (2.06mmole) of a 1M solution of lithium tri(sec-butyl)-borohydride(L-Selectride®) in THF. After stirring the solution at -75° C. for 30min, a solution of 3,5-bis(trifluoromethyl)benzyl alcohol,trifluoromethanesulfonate ester in toluene was added by cannula so thatthe internal temperature was maintained below -60° C. The resultingsolution was stirred at -75° C. for 1 hr and then between -38° C. and-50° C. for 2 hr. The solution was then poured into a mixture of 25 mLof ethyl acetate and 20 mL of saturated aqueous sodium bicarbonate, andthe layers were separated. The aqueous phase was extracted with 2×30 mLof ethyl acetate, the combined organic layers were dried over sodiumsulfate, the mixture was filtered and the filtrate concentrated invacuo. The residue was purified by flash chromatography on 130 g ofsilica eluting with 2L of 100:5 hexanes:ethyl acetate to give 0.68 g(73%) of an oil, which by ¹ H NMR is a 20:1 mixture of cis:transmorpholines.

1H NMR (CDCl₃, 400 MHz, ppm): δmajor (cis) isomer: 2.37 (td, J=12, 3.6,1H), 2.86 (app t, J=13, 2H), 3.57 (d, J=2.6, 1H), 3.63 (dq, J=11.3, 1,6,1H), 3.89 (d, J=13.3, 1H), 4.12 (td, J=11.6, 2.4, 1H), 4.40 (d, J=13.6,1H), 4.69 (d, J=2.9, 1H), 4.77 (d, J=13.6), 7.2-7.4 (m, 8H), 7.43 (s,2H), 7.55 (br d, 2H), 7.69 (s, 1H).

Step F:2-(S)-(3,5-Bis(trifluoromethyl)benzyloxy)-3-(S)-phenyl-morpholine

A mixture of 0.68 g (1.37 mmole) of4-benzyl-2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-3-(S)-phenyl-morpholineand 280 mg of 10% Pd/C in 36 mL of 97:3 ethanol:water was stirred underone atmosphere of hydrogen for 15 hr. The mixture was filtered throughCelite, the filter cake was washed generously with ethanol, and thefiltrated was concentrated in vacuo. The residue was purified by flashchromatography on 68 g of silica eluting with 1L of 33:67 hexanes:diethyl ether, then 1L of 25:75 hexanes:diethyl ether to give 0.443 g(80%) of an oil, which by 1H NMR was pure cis morpholine.

1H NMR (CDCl₃, 400 MHz, ppm): δ1.8 (br s, 1H), 3.10 (dd, J=12.5, 2.9,1H), 3.24 (td, J=12.2, 3.6, 1H), 3.62 (dd, J=11.3, 2.5, 1H), 4.04 (td,J=11.7, 3, 1H), 4.11 (d, J=2.4, 1H), 4.49 (d, J=13.5, 1H), 4.74 (d,J=2.5, 1H), 4.80 (d, J=13.3, 1H), 7.25-7.40 (m, 5H), 7.40 (s, 2H), 7.68(s, 1H). Analysis Calcd for C₁₉ H₁₇ F₆ NO₂ : C, 56.30; H, 4.23; N, 3.46; F, 28.12. Found: C, 56.20; H, 4.29 ; N, 3.34; F, 27.94.

Step G:4-(2-(N-Methylcarboxy-acetamidrazono)-2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-3-(S)-phenyl-morpholine

A solution of 2.30 g (5.7 mmol) of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-3-(S)-phenyl-morpholine, 1.13g (6.8 mmol) of N-methylcarboxy-2-chloroacteamidrazone, and 1.50 mL (8.6mmol) N,N-diisopropylethylamine in 25 mL of acetonitrile was stirred atroom temperature for 20 hours. The product, which had preciptated, wasfiltered, washed with 5 mL of ice cold acetonitrile and dried to give1.83 g of a white solid. The filtrate was concentrated in vacuo and theresidue was partitioned between 50 mL of methylene chloride and 20 mL ofwater. The layers were separated and the organic layer was dried overmagnesium sulfate. The aqueous layer was extracted with 50 mL ofmethylene chloride; the extract was dried, combined with the originalorganic layer, and the combined organics were concentrated in vacuo. Theresidue was purified by flash chromatography on 30 g of silica geleluting with 50:1:0.1 v/v/v methylene chloride/methanol/ammoniumhydroxide to afford an additional 1.09 g of product (96% total).

Mass Spectrum (FAB): m/Z 535 (M+H, 100%), 462 (16%), 291 (30%), 226(35%), 173 (25%).

¹ H NMR (CDCl₃, 400 MHz, ppm): δ2.53 (dt, J=3.5, 12.2, 1H), 2.59 (d,J=14.6, 1H), 2.94 (d, J=11.8, 1H), 3.37 (d, J=14.6, 1H), 3.58 (d,J=2.8), 1H), 3.62-3.72 (m, 1H), 3.75 (s, 3H), 4.16 (dt, J=2.2, 11.8,1H), 4.44 (d, J=13.2, 1H), 4.70 (d, J=2.8, 1H), 4.79 (d, J=13.2), 5.55(br s, 2H), 7.30-7.46 (m, 7H), 7.72 (s, 1H).

Step H:2-(S)-(3,5-Bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine

A solution of 2.89 g (5.4 mmol) of4-(2-(N-methylcarboxyacetamidrazono)-2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-3-(S)-phenyl-morpholine (from Step B) in 36 mL of xylenes washeated at reflux for 1.5 hours. The solution was cooled and concentratedin vacuo. The residue was taken up in 50 mL of 3:1 v/v hexanes/ethylacetate which caused crystallization of the product. The product wasfiltered and dried to afford 1.85 g of a solid. Recrystallization of thesolid from 30 mL of 4:1 v/v hexanes/ethyl acetate afforded 1.19 g ofpure product as a white solid. All of the crystallization liquors werecombined and concentrated in vacuo. The residue was purified by flashchromatography on 30 g of silica gel eluting with 50:1:0.1 v/v/vmethylene chloride/methanol/ammonium hydroxide to afford an additional0.69 g of a solid. Three recrystallizations from 20 mL of 4:1 v/vhexanes/ethyl acetate afforded an additional 0.39 g of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine as a white solid (58%total) (mp=156°-157° C.; designated "Form A").

Mass Spectrum (FAB): m/Z 503 (M+H), 259 (55%), 226 (40%), 160 (3%). ¹ HNMR (CDCl₃, 400 MHz, ppm): δ2.57 (app t, J=9.6, 1H), 2.87-2.97 (m, 2H),3.58-3.71 (m, 3H), 4.18 (app t, J=10.4, 1H), 4.46 (d, J=13.6), 4.68 (d,J=2.8, 1H), 4.85 (d, J=13.6, 1H), 7.30-7.45 (m, 7H), 7.64 (s, 1H), 10.40(br s, 1H), 10.73 (br s, 1H).

EXAMPLE 2 Preparation of Form B of2-(S)-(3,5-Bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine

Form A of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinewas suspended in 40 ml of deionized water and the resultant mixture wassonicated for approximately 1 minute, then stirred at room temperaturefor 48 hours. The mixture was transferred to a centrifuge tube and spunat 2,700 G for 20 minutes. The supernatant was removed and the solid wasplaced on a piece of filter paper and allowed to air-dry for 4 days.

EXAMPLE 3 Preparation of Seed Crystals of Form C of2-(S)-(3,5-Bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine

A 3.0 mg sample of Form B of2-(S)-(3,5-bis(trifluoro-methyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinewas placed in a small aluminum pan and the unsealed pan was placed in aDifferential Scanning Calorimeteric Cell (DSC) instrument. The samplewas heated at 1 ° C./min under an inert nitrogen atmosphere from ambienttemperature to 160° C., then cooled back down to room temperature. Theresulting solid is suitable for use as seed crystals in the large-scalepreparation of Form C of2-(S)-(3,5-bis(trifluoro-methyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4triazolo)methyl)-3-(S)-phenyl-morpholine.

EXAMPLE 4 Preparation of Form C of2-(S)-(3,5-Bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholine

Form A of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinewas suspended in 40 ml of deionized water and the resultant mixture wasvigorously stirred at room temperature. A few seed crystals of Form C of2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinewere added and the mixture was heated to 40° C. Stirring was continuedfor 18 hours, then the mixture was cooled to ambient temperature, andthe resultant solid was collected by filtration. The collected solid wasair-dried on a piece of filter paper for 30 minutes.

EXAMPLE 5 Typical Pharmaceutical Compositions Containing the Compound ofthe Invention

    ______________________________________                                        A: Dry Filled Capsules Containing 5 mg of Active Ingredient                   Per Capsule                                                                   Ingredient     Amount per capsule (mg)                                        ______________________________________                                        Active ingredient                                                                             5                                                             Lactose        194                                                            Magnesium stearate                                                                            1                                                             Capsule (size No. 1)                                                                         200                                                            ______________________________________                                    

The active ingredient may be reduced to a No. 60 powder and the lactoseand magnesium stearate may then be passed through a No. 60 blottingcloth onto the powder. The combined ingredients may then be mixed forabout 10 minutes and filled into a No. 1 dry gelatin capsule.

B: Tablet

A typical tablet would contain the active ingredient (5 mg),pregelatinized starch USP (82 mg), microcrystalline cellulose (82 mg)and magnesium stearate (1 mg).

C: Suppository

Typical suppository formulations for rectal administration contain theactive ingredient (0.08-1.0 mg), disodium calcium edetate (0.25-0.5 mg),and polyethylene glycol (775-1600 mg). Other suppository formulationsmay be made by substituting, for example, butylated hydroxytoluene(0.04-0.08 mg) for the disodium calcium edetate and a hydrogenatedvegetable oil (675-1400 mg) such as Suppocire L, Wecobee FS, Wecobee M,Witepsols, and the like, for the polyethylene glycol.

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various adaptations, changes, modifications,substitutions, deletions, or additions of procedures and protocols maybe made without departing from the spirit and scope of the invention.For example, effective dosages other than the particular dosages as setforth herein above may be applicable as a consequence of variations inthe responsiveness of the mammal being treated for any of theindications with the compound of the invention indicated above.Likewise, the specific pharmacological responses observed may varyaccording to and depending upon the particular active compound selectedor whether there are present pharmaceutical carriers, as well as thetype of formulation and mode of administration employed, and suchexpected variations or differences in the results are contemplated inaccordance with the objects and practices of the present invention. Itis intended, therefore, that the invention be defined by the scope ofthe claims which follow and that such claims be interpreted as broadlyas is reasonable.

What is claimed is:
 1. A polymorphic form of the compound2-(S)-(3,5-bis(trifluoromethyl)-benzyloxy)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)-3-(S)-phenyl-morpholinedesignated Form C,which is characterized by an endotherm of melting atan extrapolated onset temperature of 176°-178° C. when heated in adifferential scanning calorimetric cell at a rate of 5° C./min under anitrogen atmosphere, which is characterized by an X-ray powderdiffraction pattern with reflections at approximately: 11.5°, 12.6°,15.5°, 16.8°, 20.1°, 21.3°, 25.4° and 28.5° (2 theta), and which ischaracterized by an aqueous solubility of 2.7 μ/mL in distilled water atroom temperature.
 2. A pharmaceutical composition comprising apharmaceutically acceptable carrier and an effective amount of thepolymorphic form of claim 1.